- Email: [email protected]
neu) in lymphocytes in breast cancer
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 (LVSI +). In samples of benign tumor in 2 of 3 expression of survivin was not determined, and one was 0.015. In CTCs, isolated from peripheral blood of breast cancer patients, all 26 samples as determined by the gene expression of survivin with an average value (M±m) 0.90±0.19 (min 0.26; max 3.90). The level of expression of the control samples did not exceed 0.003. It should be noted that the maximum volume of expression obtained in samples of tumor patients with stage N +, and especially M1, on TNM classification. Any legitimate expression of survivin, depending on the size of the tumor had been received. In patients, receiving chemotherapy observed average expression of survivin gene, but never approached the indicators of control. Conclusions: Determination of expression of the gene survivin in primary tumor and the CTCs may be treated to one of the most promising markers of tumor progression and monitoring of breast cancer therapy. No conflict of interest. 485 WWP2 controls endothelial permeability by regulating the RAP1–RADIL signaling pathway F. Renzi1 , M.F. Baietti1 , L. Abbasi1 , K. Gevaert2 , A. Sablina1 . 1 VIB, center of human genetics, Leuven, Belgium, 2 VIB, center of human genetics, Gent, Belgium Background: The small GTPase RAP1 plays a crucial role in endothelial functioning, suggesting its potential contribution in the regulation of cancer cell extravasation. The RAP1 downstream effector RADIL is involved in the formation of endothelial cell junction and control of endothelial permeability. Our previous results revealed that monoubiquitination of the RAS-like GTPases alters their binding to specific downstream effectors, thus promoting particular signaling pathways. In this study, we investigate the role of reversible ubiquitination in regulation RAP1 signaling and function. Material and Methods: Mass spectrometry analyses identified WWP2 as potential interactor of RAP1. The interaction between RAP1 and WWP2 was further confirmed by reciprocal immunoprecipitations and co-localization analysis of the two proteins. We also assessed the effect of WWP2 expression on status of RAP1 ubiquitination. Endothelial permeability was measured by quantifying the amount of FITC-dextran passing through a monolayer of Human Umbilical Vein Endothelial Cells. Results: We found that RAP1 undergoes monoubiquitination within its effector binding domain. Our results implicated the E3 ubiquitin ligase WWP2 in control of RAP1 monoubiquitination. Importantly, depletion of WWP2 negatively affects the binding of RAP1 to its effector RADIL, resulting in increased endothelial permeability. Furthermore, increased endothelial permeability triggered by loss of WWP2 facilitates extravasation of cancer cells through a monolayer of endothelial cells. Altered endothelial permeability caused by WWP2 depletion could facilitate the spreading of metastatic cancer cells. Recently several anti-cancer approaches have been focused on the inhibition of E3 ligases. Specifically, it has been proposed that inhibition of WWP2 can suppress tumor growth by increasing levels of the tumor suppressor PTEN. However, our data indicate that suppression of WWP2 in stroma may increase metastatic spreading. Conclusion: WWP2-mediated RAP1 monoubiquitination plays a key role in the regulation of endothelial function and extravasation of cancer cells by promoting the interaction between RAP1 and its downstream effector RADIL. Understanding the mechanisms that affect tumor extravasation can lead to the development of specific therapies that aim to reduce the metastatic potential of cancer cells. No conflict of interest. 486 Expression of survivin gene (BIRC5) and ErbB-2 (Her-2/neu) in lymphocytes in breast cancer Y. Shlyakhtunou1 , V. Semenov2 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, Oncology, Vitebsk, Belarus, 2 Vitebsk State Order of Peoples’ Friendship Medical University, Infectious Diseases, Vitebsk, Belarus It is now known that the presence of tumor infiltrating lymphocytes (TILs) is an indirect indication of the active anti-tumor immunity and combined with improved prognosis in patients, breast cancer (breast cancer) suffering from resectable cancer. According to the literature, patients with a high content of TILs holding one chemotherapy alone was associated with a 5-year disease-free survival rate of 91%. Adding Anita-Her-2 directional drug trastuzumab in this group did not improve outcomes. On the other hand, it was observed nonsignificant decrease in 5-year disease-free survival to 80%. The explanation of this phenomenon yet. Perhaps this is due to antigen-presenting mechanism of lymphocytes themselves. Objective: To study the expression of the gene survivin (BIRC5), and ErbB-2 (Her-2/neu) in lymphocytes infiltrating breast carcinoma tissue, and in peripheral blood lymphocytes of patients suffering from breast cancer. Materials and Methods: After homogenization of frozen tumor specimen isolated lymphocytes (CD45 +) by separation. In the same way the lymphocytes isolated from the peripheral blood. Using real-time PCR Gene
S105
expression was investigated BIRC5 ErbB-2 and lymphocytes (phenotype) 16 samples of primary invasive ductal breast carcinomas and in 26 samples of peripheral blood of patients suffering from breast cancer. Results: B lymphocytes isolated from all carcinoma samples was determined by gene expression BIRC5 and ErbB-2 with a mean value (M±ms) for survivin 2.00±1.29 (min 0.003; max 4.283) and 0.57±0.29 (min 0.024; max 1.459) for the Her-2/neu. B lymphocytes isolated from peripheral blood, in all the 26 samples was determined by the expression of ErbB-2 (Her-2/neu) with the average value (M±ms) 0.661±0.521 (min 0.259; max 1.405). In 19 samples, representing 73% of the expression of survivin gene was determined with the average value (M±ms) 0.248±0.171 (min 0.035; max 0.488). The 7 samples (27%) yielded negative results BIRC5 gene expression. Conclusion: The findings suggest that a sort of “tension” cellular immunity directly in the tumor tissue and the bloodstream. The wide range of indicators may suggest a different immune response of the organism to the tumor. This study indicates a possible predictive value of TILs as a biomarker that can identify a group of patients who are highly sensitive to chemotherapy and do not require the addition of trastuzumab. However, this hypothesis requires confirmation in randomized trials. No conflict of interest. 487 Identification and exploitation of collateral vulnerabilities to transcription and RNA processing inhibitors in tumor subtypes L. Frischknecht1 , Y. Christinat1 , C. Britschgi1 , W. Krek1 . 1 Molecular Health Sciences, Biology, Zurich, ¨ Switzerland Introduction: Genome instability is a general feature of cancer cells. Frequently, these alterations cause deletions of loci containing tumor suppressor genes and concurrently lead to hemizygous copy loss of flanking genes. The latter, when affecting genes acting in functionally essential processes, generates cancer specific vulnerabilities that could be exploited therapeutically. Material and Method: Bioinformatics analysis and data integration was performed on the Achilles, CCLE and TCGA databases (Broad Institute and NIH) with a focus on the URI1 onco-chaperone network. Drug response curves were performed on a panel of different representative cancer cell lines using PrestoBlue® cell viability reagent. CRISPR interference and activation was used to up- or down-regulate relevant genes to test causality in mediating collateral vulnerability. Results and Discussion: URI1 is a member of the prefoldin chaperone complexes with established oncogenic properties in multiple cancers. Analysis of the Achilles database (http://www.broadinstitute.org/achilles), containing information from loss-of-function screens in 216 cancer cell lines, revealed an URI1 onco-chaperone gene network comprised of four subnetworks involved in transcription, RNA processing, nuclear transport and protein degradation that demonstrated very similar survival properties as URI1. Further in silico analysis revealed that the dependency of cancer cells for their survival on genes in this new network correlated with copy number alterations of five of these genes as well as on the transcriptional burden of cancer cells as assessed by the copy number of a list of established driver oncogenes. Chemical inhibition of components of the individual subnetworks validated differential sensitivity of cancer cells as a function of hemizygous loss of network genes and oncogene copy number. The copy number changes of the five genes correlating with network sensitivity are driven by amplification or deletion of neighboring oncogenes and tumor suppressor genes respectively. The strongest correlation was observed with the copy number of POLR2E, a shared subunit of all three nuclear RNA polymerases and established chaperone target of URI1. POLR2E shows frequent hemizygous losses in melanoma, as a flanking gene of the tumor suppressor STK11. Melanoma cell lines having only one copy of POLR2E displayed increased sensitivity to RNA polymerase inhibition. Conclusion: Collateral hemizygous deletions of ‘housekeeping-genes’ that comprise the URI1 onco-chaperone network might be an interesting novel therapeutic target especially in cancers characterized by increased demand on the functions of the identified gene subnetworks. No conflict of interest. 488 ERK5 is required for pro-tumour macrophage activation E. Giurisato1 , W. Vermi2 , C. Tournier1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 2 University of Brescia, Medicina Molecolare e Traslazionale, Brescia, Italy Background: A number of transcription factors have been implicated in the rearrangement of the transcriptional profile that sustains the reprogramming of tumour-associated macrophages (TAMs) to facilitate carcinoma development. In contrast, we have a very limited understanding of the signalling mechanisms that governs this process in response to tumour-derived signals. The extracellular-regulated protein kinase 5 (ERK5) is a unique mitogenactivated protein kinase (MAPK) which has previously been linked to the pathophysiology of cancer.
S105
expression was investigated BIRC5 ErbB-2 and lymphocytes (phenotype) 16 samples of primary invasive ductal breast carcinomas and in 26 samples of peripheral blood of patients suffering from breast cancer. Results: B lymphocytes isolated from all carcinoma samples was determined by gene expression BIRC5 and ErbB-2 with a mean value (M±ms) for survivin 2.00±1.29 (min 0.003; max 4.283) and 0.57±0.29 (min 0.024; max 1.459) for the Her-2/neu. B lymphocytes isolated from peripheral blood, in all the 26 samples was determined by the expression of ErbB-2 (Her-2/neu) with the average value (M±ms) 0.661±0.521 (min 0.259; max 1.405). In 19 samples, representing 73% of the expression of survivin gene was determined with the average value (M±ms) 0.248±0.171 (min 0.035; max 0.488). The 7 samples (27%) yielded negative results BIRC5 gene expression. Conclusion: The findings suggest that a sort of “tension” cellular immunity directly in the tumor tissue and the bloodstream. The wide range of indicators may suggest a different immune response of the organism to the tumor. This study indicates a possible predictive value of TILs as a biomarker that can identify a group of patients who are highly sensitive to chemotherapy and do not require the addition of trastuzumab. However, this hypothesis requires confirmation in randomized trials. No conflict of interest. 487 Identification and exploitation of collateral vulnerabilities to transcription and RNA processing inhibitors in tumor subtypes L. Frischknecht1 , Y. Christinat1 , C. Britschgi1 , W. Krek1 . 1 Molecular Health Sciences, Biology, Zurich, ¨ Switzerland Introduction: Genome instability is a general feature of cancer cells. Frequently, these alterations cause deletions of loci containing tumor suppressor genes and concurrently lead to hemizygous copy loss of flanking genes. The latter, when affecting genes acting in functionally essential processes, generates cancer specific vulnerabilities that could be exploited therapeutically. Material and Method: Bioinformatics analysis and data integration was performed on the Achilles, CCLE and TCGA databases (Broad Institute and NIH) with a focus on the URI1 onco-chaperone network. Drug response curves were performed on a panel of different representative cancer cell lines using PrestoBlue® cell viability reagent. CRISPR interference and activation was used to up- or down-regulate relevant genes to test causality in mediating collateral vulnerability. Results and Discussion: URI1 is a member of the prefoldin chaperone complexes with established oncogenic properties in multiple cancers. Analysis of the Achilles database (http://www.broadinstitute.org/achilles), containing information from loss-of-function screens in 216 cancer cell lines, revealed an URI1 onco-chaperone gene network comprised of four subnetworks involved in transcription, RNA processing, nuclear transport and protein degradation that demonstrated very similar survival properties as URI1. Further in silico analysis revealed that the dependency of cancer cells for their survival on genes in this new network correlated with copy number alterations of five of these genes as well as on the transcriptional burden of cancer cells as assessed by the copy number of a list of established driver oncogenes. Chemical inhibition of components of the individual subnetworks validated differential sensitivity of cancer cells as a function of hemizygous loss of network genes and oncogene copy number. The copy number changes of the five genes correlating with network sensitivity are driven by amplification or deletion of neighboring oncogenes and tumor suppressor genes respectively. The strongest correlation was observed with the copy number of POLR2E, a shared subunit of all three nuclear RNA polymerases and established chaperone target of URI1. POLR2E shows frequent hemizygous losses in melanoma, as a flanking gene of the tumor suppressor STK11. Melanoma cell lines having only one copy of POLR2E displayed increased sensitivity to RNA polymerase inhibition. Conclusion: Collateral hemizygous deletions of ‘housekeeping-genes’ that comprise the URI1 onco-chaperone network might be an interesting novel therapeutic target especially in cancers characterized by increased demand on the functions of the identified gene subnetworks. No conflict of interest. 488 ERK5 is required for pro-tumour macrophage activation E. Giurisato1 , W. Vermi2 , C. Tournier1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 2 University of Brescia, Medicina Molecolare e Traslazionale, Brescia, Italy Background: A number of transcription factors have been implicated in the rearrangement of the transcriptional profile that sustains the reprogramming of tumour-associated macrophages (TAMs) to facilitate carcinoma development. In contrast, we have a very limited understanding of the signalling mechanisms that governs this process in response to tumour-derived signals. The extracellular-regulated protein kinase 5 (ERK5) is a unique mitogenactivated protein kinase (MAPK) which has previously been linked to the pathophysiology of cancer.